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D A Deranleau

Publications and source records attributed to D A Deranleau.

At least 19 recordsLinked to original sources
PubMed1995

ADP-ribosylation of Rho enhances actin polymerization-coupled shape oscillations in human neutrophils.

Stimulated neutrophils exhibit coordinated sinusoidal oscillations in filamentous actin content and cellular shape. We investigated the effect of inhibition of the small G protein Rho on neutrophil actin polymerization, shape changes and oscillations using a genetically engineered toxin that enters cells and selectively ADP-ribosylates endogenous Rho. This treatment increased the amplitudes and frequencies of shape oscillations and duration of the oscillating transient. However, it had no effect on the initial actin polymerization and shape changes induced by N-formyl-Met-Leu-Phe. Regulation of these oscillations may be important for the control of neutrophil motility.

Actins↗
PubMed1991

Shape changes, exocytosis, and cytosolic free calcium changes in stimulated human eosinophils.

Essentially pure preparations of normal density eosinophils obtained from patients with hypereosinophilic syndrome (HES) were stimulated with complement factor 5a (C5a), platelet-activating factor (PAF), FMLP and neutrophil-activating peptide (NAP-1/IL-8). Three responses were studied, the transient rise in cytosolic free calcium concentration ([Ca2+]i) (derived from indo-1 fluorescence), shape changes (measured by laser turbidimetry), and exocytosis of eosinophil peroxidase (EPO) (assessed by H2O2/luminol-dependent chemiluminescence). Responses were obtained with all four agonists, but C5a and PAF were by far more potent than FMLP and NAP-1/IL-8, which induced only minor effects. Pretreatment of the cells with pertussis toxin attenuated [Ca2+]i changes, EPO release and, to a lesser extent, shape changes, indicating that GTP-binding proteins of Gi-type are involved in receptor-dependent signal transduction processes leading to these responses. A clear dissociation was observed in the control of the shape change response and EPO exocytosis. The shape change was not affected by Ca2+ depletion or treatment with the protein kinase inhibitor staurosporine, but exocytosis was prevented by Ca2+ depletion and markedly enhanced by staurosporine. The activation of the contractile system, leading to shape changes and motility, thus appears to be independent of the classical signal transduction pathway involving phospholipase C, a [Ca2+]i rise and protein kinase C activation. Exocytosis is, as expected, Ca2+ dependent and appears to be under a negative control involving protein phosphorylations.

Alkaloids↗
PubMed1990

Corresponding oscillations in neutrophil shape and filamentous actin content.

Human neutrophils pretreated with 17-hydroxywortmannin responded to the chemotactic agonist formyl-Met-Leu-Phe with a transient doubling in filamentous actin content which was characterized by prominent oscillations. These oscillations closely matched transient oscillations in suspension turbidity measured in parallel. The experimental data could be simulated using A----B----C stochastic series kinetic models with an oscillating intermediate species (B), allowing quantitative comparison of the frequencies of the oscillations (0.092 +/- 0.006 and 0.094 +/- 0.004 Hz) and the overall reaction rate constants for actin mobilization and turbidity changes (0.11 +/- 0.02 and 0.14 +/- 0.03 s-1, respectively). The total cell volume remained constant, indicating that stimulus-induced extension of lamellipods reduces the body volume by an amount proportional to the mass displaced outward. Light scattering theory predicts that a decrease in body size decreases the turbidity and that fluctuations in body size due to lamellipod extension and retraction cycles like those exhibited by crawling neutrophils result in turbidity oscillations (lamellipods scatter very little by comparison to the cell body, and both aggregation and degranulation were absent). The experiments thus suggest that the cyclic variations in F-actin content are correlated with periodic fluctuations in lamellipod size. The available evidence appears to be consistent with the hypothesis that actin polymerization provides the main driving force for lamellipod extension and that depolymerization causes lamellipod retraction.

Actins↗
PubMed1989

Respiratory burst oscillations in human neutrophils and their correlation with fluctuations in apparent cell shape.

Neutrophils pretreated with phorbol 12-myristate 13-acetate (1-10 nM) and stimulated with low concentrations of chemotactic agonists (1-10nM) exhibited a marked increase in respiratory burst activity that was characterized by regular oscillations. These were accompanied by parallel oscillations in turbidity having the same phase and period. Four different agonists, f-Met-Leu-Phe, complement fragment C5a, platelet-activating factor, and leukotriene B4, induced virtually identical oscillations, with mean periods of 7.9 +/- 0.6 s (respiratory burst) and 7.9 +/- 0.8 s (turbidity) at 37 degrees C. No burst oscillations were observed at high agonist concentrations (50-100 nM) unless the fungal metabolite 17-hydroxywortmannin was added prior to stimulation. In the absence of phorbol 12-myristate 13-acetate, the respiratory burst activity was inhibited by 17-hydroxywortmannin, the protein kinase C inhibitor staurosporine, and calcium depletion, while agonist-dependent turbidity changes including the oscillations were unaffected. Turbidity changes reflect corresponding changes in cell size and/or shape, suggesting that cyclic alterations in morphology such as lamellipod extension and retraction physically affect the catalytic efficiency of the membrane-bound burst enzyme NADPH-oxidase. The oscillations appear to be controlled via receptor-dependent activation mechanisms which do not involve PKC activation or the rise in internal calcium presumably derived from phospholipase C activation.

Androstadienes↗
PubMed1989

[Properties and activation mechanism of neutrophilic leukocytes].

The mechanism of neutrophil activation by chemotactic receptor agonists was studied by monitoring stimulus-induced changes in the cytosolic free calcium concentration and hydrogen peroxide formation during the respiratory burst. Two receptor-dependent signal transduction sequences were identified. One sequence depends on calcium, phospholipase C and protein kinase C, and is rate limiting, while the other is comparatively fast and appears to be calcium-independent. The present studies indicate that both sequences must act in concert to transduce receptor-mediated signals and to activate the NADPH oxidase.

Animals↗
PubMed1988

Real-time models of morphogenetic processes.

Morphological transformations are often describable by simple series kinetic models, A----B, A----B----C, etc., which allow assessment of the rates of interconversion of the distinguishable shapes or forms present and their probabilities of occurrence at various points in time, thus providing a means for kinetic comparisons with biochemical measurements of the molecular-level reactions that cause the transformations. When changes in cell morphology are followed turbidimetrically, the real-time progress curves can be simulated by fitting the data to a form of Beer's law for scattering by mixtures in which the species concentrations change with time in accordance with the chosen kinetic scheme. Because many even relatively large cells are mostly water, classical light scattering theory can be used to interpret the turbidimetric data in terms of simple geometrical models of average cell size and shape suggested by microscopic examination. Two examples are briefly considered, the stimulus-induced changes in blood platelet shape and apparent size and their correlation with cytosolic-free calcium, and apparent swimming motion exhibited by neutrophils in suspension.

Blood Platelets↗
PubMed1987

Kinetic models of cell shape changes and other optically-detected responses to stimulation.

Certain cellular responses to stimulation can be described as a continuous series of virtually infinite but real transient states generated by reactions occurring at the molecular level within the cell. While any particular state can in principle be isolated and examined by using appropriate methods to stop or freeze the reaction, adjacent or nearby states will generally be indistinguishable from one another by kinetic means. In favorable cases, however, the progress curves can be fitted to comparatively very simple kinetic models involving a limited number of steps, which accurately describe the real-time response within the limitations of the experimental setup. The simple series models have their origins in the continuous myriad-state or "microscopic" series description, and the observable or "macroscopic" kinetic rate constants are statistically related to the rate constants describing the transitions between the real states of the ongoing response. This indicates that different aspects of stimulus-response coupling, e.g., shape changes, alterations in cytosolic calcium levels and so forth, can be compared in a self-consistent fashion by modeling the individual responses in terms of simple parallel series models.

Blood Platelets↗
PubMed1987

The onset of the respiratory burst in human neutrophils. Real-time studies of H2O2 formation reveal a rapid agonist-induced transduction process.

A real-time study of the initiation of the respiratory burst in human neutrophils was made. The cells were stimulated with fMet-Leu-Phe (fMLP) C5a, platelet-activating factor, leukotriene B4, phorbol myristate acetate (PMA), or ionomycin, and H2O2 production was determined by chemiluminescence. Identical average onset times (2.4 s) and closely comparable values for the apparent first-order rate constant (kapp) for the induction of NADPH-oxidase activity (0.21-0.29 s-1) were obtained following stimulation with fMLP, C5a, platelet-activating factor, or leukotriene B4, suggesting that different agonists act through a common transduction sequence. Much longer onset times and lower kapp values were obtained upon stimulation with PMA or ionomycin. Pretreatment with PMA consistently shortened the onset time of the neutrophil's responses to agonists by about 1 s. When H2O2 production was initiated with PMA, a subsequent stimulation with the agonist fMLP elicited an immediate response (onset time less than 0.2 s) which preceded further changes in fura-2-detected [Ca2+]i. The results are consistent with a mechanism in which agonist signals appear to be transduced by two sequences acting in concert--a rate-limiting one liberating Ca2+ and diacylglycerol and turning on the Ca2+/phospholipid-dependent enzyme protein kinase C, and an essentially instantaneous one which does not appear to require further changes in cytosolic Ca2+.

Ethers↗
PubMed1987

Oscillatory motion in human neutrophils responding to chemotactic stimuli.

Human neutrophils treated with the secretion inhibitor 17-hydroxywortmannin were stimulated with fMLP, C5a, PAF or LTB4, and the ensuing shape change was studied. The cells rapidly extended lamellipodia and showed regular oscillatory behaviour. The oscillations were observed in both light transmission and 90 degrees light scattering, had the same frequency in each case, and disappeared within 30-50 seconds. Light scattering theory suggests that they reflect rhythmic changes in the shape and/or size of the chemotactically stimulated cells, possibly related to crawling or swimming movements associated with migration.

Androstadienes↗
PubMed1987

Chemiluminescence detection of H2O2 produced by human neutrophils during the respiratory burst.

A sensitive luminol-dependent chemiluminescence assay for H2O2 was developed for the indirect determination of the transient changes in NADPH oxidase activity associated with the respiratory burst of human neutrophils. A relatively large, controlled amount of horseradish peroxidase was used in combination with added luminol to rapidly remove and simultaneously detect H2O2 as soon as it is formed, thus preventing its accumulation during burst activity and minimizing the effects of side reactions. Cell-derived myeloperoxidase and possibly catalase were inhibited with 90 microM sodium azide to maintain the total catalytic activity toward H2O2 at a constant level. Chemiluminescence measurements of the respiratory burst activity of human neutrophils stimulated with N-formyl-Met-Leu-Phe (fMLP) were in good agreement with measurements made using an established fluorometric assay based on similar principles (P. A. Hyslop and L. A. Sklar (1984) Anal. Biochem. 141, 280-286). In contrast to fluorometry, the chemiluminescence progress curves reflect the instantaneous rather than the integrated levels of H2O2 at any time and are thus a more direct measure of the activity of the NADPH oxidase. This advantage, as well as higher signal-to-noise ratios and greater inherent sensitivity, distinguishes chemiluminescence as a means of following burst activity. The onset of fMLP-stimulated H2O2 generation was detectable by chemiluminescence within 2 s of stimulation (as opposed to more than double this time by fluorometry), showing that high sensitivity is an important consideration in evaluating respiratory burst kinetics. In contrast to fMLP stimulation, longer and concentration-dependent onset times were observed when phorbol myristate acetate was used as a stimulus.

Horseradish Peroxidase↗
PubMed1986

Calcium fluxes and calcium buffering in human neutrophils.

Neutrophils loaded with the calcium indicator quin-2 and challenged with the ionophore ionomycin or the chemotactic peptide fMet-Leu-Phe were examined in the light of a theory that relates time-dependent changes in the fluorescence of the indicator to cytosolic calcium fluxes and levels. The cytosolic binding capacity was estimated from the theory to be 1.5 +/- 0.6 X 10(8) sites/cell (0.76 mM based on a cell volume of 330 micron 3, irrespective of water content and the distribution of sites), each site having an apparent average single class dissociation constant of 0.55 +/- 0.2 microM. Some 20% of the total available cytosolic calcium sites of the normal resting cell appear to be occupied when no quin-2 is present. In a calcium-free medium, the amount of calcium released by fMet-Leu-Phe from storage pool locations that are distinct from the cytosolic sites is sufficient to further raise the cytosolic site occupancy level to 50%, at which point the calcium buffering capacity of the cytosol is maximal. In a calcium-containing medium, however, simultaneous influx from the outside appears to supply enough additional calcium to saturate most of the remaining sites. The combined initial rate of storage pool calcium release plus influx through the plasma membrane was roughly twice the initial rate at which calcium was released from storage locations alone, suggesting that stimulus-induced influx from the outside may be comparable in importance to storage pool mobilization in determining physiological calcium levels in stimulated cells.

Aminoquinolines↗
PubMed1986

Stochastic response of human blood platelets to stimulation of shape changes and secretion.

Stopped-flow turbidimetric data indicate that platelets stimulated with low levels of thrombin undergo a shape transformation from disc to "sphere" to smaller spiny sphere that is indistinguishable from the shape change induced by ADP through different membrane receptor sites and a dissimilar receptor trigger mechanism. Under conditions where neither secretion nor aggregation occur, the extinction coefficients for total scattering by each of the three platelet forms are independent of the stimulus applied, and both reaction mechanisms can be described as stochastic (Poisson) processes in which the rate constant for the formation of the transient species is equal to the rate constant for its disappearance. This observation is independent of the shape assignment, and as the concentration of thrombin is increased and various storage organelles secrete increasing amounts of their contents into the external medium, the stochastic pattern persists. Progressively larger decreases in the extinction coefficients of the intermediate and final platelet forms, over and above those that reflect shape alterations alone, accompany or parallel the reaction induced by the higher thrombin concentrations. The excess turbidity decrease observed when full secretion occurs can be wholly accounted for by a decrease in platelet volume equal in magnitude to the fraction of the total platelet volume occupied by alpha granules. Platelet activation, as reported by the whole body light scattering of either shape changes alone or shape changes plus parallel (but not necessarily also stochastic) alpha granule secretion, thus manifests itself as a random series of transient events conceivably with its origins in the superposition of a set of more elementary stochastic processes that could include microtubule depolymerization, actin polymerization, and possibly diffusion. Although the real nature of the control mechanism remains obscure, certain properties of pooled stochastic processes suggest that a reciprocal connection between microtubule fragmentation and the assembly of actin-containing pseudopodal structures and contractile elements--processes that may exhibit reciprocal requirements for calcium--might provide a hypothetical basis for a rate-limiting step.

Adenosine Diphosphate↗
PubMed1984

Human blood platelet secretion: optical multichannel analyzer measurements using acriflavine as a release indicator.

Blood platelets preloaded with the fluorescent amine acriflavine release the trapped fluorophore after stimulation with thrombin or the divalent cation ionophore A23187. Release was detected by an increase in acriflavine fluorescence, which is otherwise strongly quenched in the platelet, by using an optical multichannel analyzer to monitor the spectral and temporal reaction parameters. The secretion of [14C]serotonin and acriflavine is well correlated, suggesting that acriflavine, like serotonin and the closely related fluorescent drugs mepacrine and acridine orange, is accumulated in and released from platelet dense bodies. Acriflavine secretion at 37 degrees C in the absence of external calcium is characterized by a short delay, followed by a rapid biphasic increase in fluorescence that implies at least a three-stage secretory process. For saturating levels of thrombin the delay was 1.5 s and release was 90% complete within 6-7 s. The delay could not be shortened by prestimulation under conditions that induce shape changes but not release, i.e., with ADP, arachidonic acid, or low levels of thrombin or A23187. Acriflavine secretion induced by A23187 was similar but less effective; the reaction was slower, the yield was smaller, and, in contrast to thrombin, the longer lag period could be significantly shortened by prestimulation.

Acriflavine↗
PubMed1983

Optimal efficiency of human platelet shape changes.

The light scattering extinction coefficients and kinetic rate constant(s) for the disc to sphere and the sphere to (smaller) 'spiny sphere' shape changes induced in platelets by saturating amounts of ADP are compared at 31, 37 and 41 degrees C. Platelets exhibit optimum efficiency at 37 degrees C, as judged by not only the rates of the shape changes but also by alterations in the overall sizes and shapes of the three platelet forms. Unstimulated (discoid) platelets appear to be flatter at 37 degrees C, the disc to sphere reaction appears to be impaired at other than 37 degrees C, and the pseudopodia which characterize the final spiny sphere may be more prominent at 41 degrees C.

Blood Platelets↗
PubMed1982

Transient kinetics of the rapid shape change of unstirred human blood platelets stimulated with ADP.

Unstirred (isotropic) suspensions of human blood platelets stimulated with ADP in a stopped-flow laser turbidimeter exhibit a distinct extinction maximum during the course of the classical rapid conversion of initially smooth flat discoid cells to smaller-body spiny spheres. This implies the existence of a transient intermediate having a larger average light scattering cross section (extinction coefficient) than either the disc or the spiny sphere. Monophasic extinction increases reaching the same final value were observed when either discoid or spiny sphere platelets were converted to smooth spheres by treatment with chlorpromazine, and sphering of discoid cells was accompanied by a larger total extinction change than the retraction of pseudopods by already spherical cells. These and other results suggest that the ADP-induced transient state represents platelets that are approximately as "spherical" as the irregular spiny sphere but lack the characteristic long pseudopods and as a consequence are larger bodied. Fitting the ADP progress curves to the series reaction A leads to B leads to C by means of the light scattering equivalent of the Beer-Lambert law yielded scattering cross sections that are consistent with this explanation. The rate constants for the two reaction steps were identical, indicating that ADP activation corresponds to a continuous random (Poisson) process with successive apparent states "disc," "sphere," and "spiny sphere," whose individual probabilities are determined by a single rate-limiting step.

Adenosine Diphosphate↗